Phage display is a well-established tool for affinity-based selection of polypeptides. In a typical phage display selection, a library of polypeptides is genetically fused to the terminus of one of the coat proteins of the filamentous phage M13. The phage particle provides a physical link between each polypeptide member of the library and the gene that encodes it. The phage library can then be affinity-selected, or panned, for those members of the library that bind to a desired target molecule. The library is mixed with the target, unbound phage particles are washed away, and the remaining phage eluted and amplified by culturing in E. coli cells.
Although the display of foreign polypeptides has been accomplished with each of the coat proteins of M13, pIII and pVIII are by far the most common fusion partners. pIII is a 42 kD minor coat protein that is responsible for phage infection into E. coli. Each phage particle contains up to five copies of the pIII protein on its surface, gathered at one end of the phage. PVIII is the major coat protein of the phage; thousands of copies of pVIII (molecular weight 5 kD) are arranged in an orderly fashion around the single-stranded viral genome to comprise the phage capsid. In addition to pIII, M13 has three other minor coat proteins: pVI, a 12 kD protein, and pVII and pIX, which are short proteins (33and 32 amino acids, respectively) that are involved in initiation of assembly and maintenance of stability. Five copies of the pVI protein lie at the same end of the phage as pIII, while five copies each of pVII and pIX reside at the opposite end of the phage.
While phage libraries displaying fusions to pIII and pVIII have proven productive in many cases, the polypeptides displayed by phage are subject to certain biological constraints. For instance, most peptides of eight or more amino acids in length do not display well as fusions to pVIII. In addition, polypeptides that interact with the phage protein itself or otherwise affect the expression, incorporation, or activity of pIII or pVIII will be under-represented in the library, because the phage that display them will not grow well. Finally, because pIII is a rather large protein, access of pIII-displayed polypeptides to certain target sites (deep, narrow crevices on a protein surface, for instance), or the correct assembly of polypeptides that function best in multimeric form, might be sterically hindered. Thus, selections from phage libraries that exploit other coat proteins—which have different structures and biological functions and thus might be expected to impose different constraints on displayed polypeptides would help to ensure that a maximum amount of sequence diversity is searched. In proof of concept experiments, it has been shown that pVII and pIX can be used for the display of both antibody fragments and peptides. These results were especially noteworthy since earlier work had suggested that fusions of polypeptides to the N-termini of pVII and pIX render these coat proteins non-functional.
The display of foreign polypeptides on phage is accomplished through the use of phage, phagemid, or hybrid vectors. With phage vectors, the gene of interest is introduced into the phage genome as an in-frame fusion with the native coat protein gene. These vectors propagate independently as fully functional phage and display multiple copies of the foreign polypeptide. Phagemid vectors, in contrast, are plasmids that contain a phage origin of replication and packaging signal in addition to a bacterial origin of replication. Phagemids carry the gene of interest fused to a recombinant copy of the coat protein gene and, upon rescue with a helper phage, are packaged into progeny virus with the displayed polypeptide incorporated into the phage coat. The requirement for helper phage causes phagemid vectors to be more labor-intensive than phage vectors, and complicates efforts to quantitate the number of phage that are present in any given sample. Furthermore, since phage particles can draw upon both wild-type coat proteins and fusion coat proteins for assembly, some proportion of the resultant phage will not display the polypeptide sequence of interest, resulting in low display efficiency. Hybrid vectors resemble phage vectors in that the fusion protein is carried in the phage genome and no helper phage are needed, but they also resemble phagemid systems in that the genome also carries a wild-type copy of the fusion protein. Previous reports of pIX phage display describe fusions in the context of phagemid vectors; display of polypeptides on pIX from a hybrid or phage vector has not previously been reported. Display of polypeptides on pVII from a phage vector has recently been reported.
There is a need for providing synthetic non-antibody peptide or protein libraries and methods that simultaneously deliver the critical elements of human therapeutic peptides and proteins of high affinity and activity, high productivity, good solution properties, and a propensity of low immune response when administered in man. There is a further need to increase the efficiency of non-antibody peptide or protein isolation from synthetic libraries, relative to current methods, to reduce the resource costs of non-antibody peptide or protein discovery and accelerate delivery of non-antibody peptides or proteins for biological evaluation. The libraries and methods of this invention meet these needs by coupling comprehensive design, assembly technologies, and phage pIX Peptide or protein display.